It is known that troponin is a myofibrillar protein complex composed of troponin I, troponin T, and troponin C, and interacts with actin and myosin to contribute in the regulation of muscle contraction by calcium ions. More particularly, when the impulse reaches the level of the motor endplate of the muscle, action potential is generated and transmitted to the sarcoplasmic reticulum. Then, calcium ions are released into the cytosol, and bind to troponin C. A conformational change of the troponins I, T, and C complex occurs by enhancing the interaction between troponin I and troponin C, and it enables muscle contraction by the actin-myosin interaction.
When cardiac muscle is irreversibly damaged, the cardiac troponin that is released appears in the blood stream. Three subunits, i.e., cardiac troponin I, cardiac troponin C, and cardiac troponin T, alone or as a complex (cardiac troponin) consisting of two or three different subunits, exist in the blood.
A blood sample (serum, plasma, or whole blood) is commonly used to evaluate various cardiac troponins. However, such a selection may be limited by the method used. For example, it is known that a serum is an inappropriate biological sample in a method for rapidly evaluating cardiac troponin, and that whole blood complicates the implementation of a quantitative assay. In an immunological assay using heparinized plasma or heparinized whole blood, even when the performance of the method used is very high, unreliable results are often obtained. In general, this problem occurs when the concentration of cardiac troponin in plasma is not very high (non-patent literature 1). Actually, it is known that the presence of heparin in a blood sample interferes with various immunological assays and affects the measurement results. Because of this, the clinical diagnosis of a doctor may be modified.
An EDTA blood collection tube is also commonly used as an anticoagulant, EDTA plasma or EDTA whole blood may cause unreliable results. In general, cardiac troponin I forms a complex with cardiac troponin C and/or cardiac troponin T in the presence of calcium ions. However, in the presence of EDTA, since calcium ions are chelated, the complex containing cardiac troponin I decomposes. As a result, it is known that EDTA plasma or EDTA whole blood affects an immunological assay due to a change in the form of cardiac troponin I in blood (non-patent literature 2).
With respect to a method for avoiding the influence of interfering substances in a specimen in a cardiac troponin assay, an immunological measuring method characterized in that it is carried out using a biological sample containing heparin in the presence of hexadimethrine bromide (polybrene) is disclosed (patent literature 1). This relates to a method for avoiding the interference caused by heparin, and this literature does not refer to EDTA.
Further, it is disclosed that the addition of a divalent cation to cardiac troponin is effective in stabilizing the cardiac troponin complex (patent literatures 2, 3, and 4).
For example, patent literature 2 discloses a composition containing calcium chloride or magnesium chloride at a concentration of 100 μmol/L to 100 mmol/L, as a buffer for a stable composition of troponin complex, and an embodiment containing 2 mmol/L calcium chloride, as a preferable composition (paragraph [0013]). However, patent literature 2 does not refer to the concentration of calcium chloride or magnesium chloride in a reaction solution during the troponin assay (i.e., when the immunological complex is formed). Therefore, it is not disclosed nor suggested in patent literature 2 that a high concentration should be maintained in the reaction solution for the immunological measurement of cardiac troponin, and that an accurate value sometimes cannot be obtained at a low concentration (for example, 2 mmol/L) depending on the type of specimen.
Patent literature 3 discloses that, as a preferable composition of stabilized troponin I, the concentration of calcium chloride or magnesium chloride is 0.01 mmol/L to 10 mmol/L. Further, it is disclosed in Example 14 that calcium chloride is added to a serum or plasma so as to become a final concentration of 6 mmol/L before carrying out an antigen-antibody reaction, the resulting solution is incubated at room temperature for 2 hours followed by 4° C. overnight, and the incubated sample is diluted with a metal-free assay buffer via a dilution factor of 2 to 256 (maximum). In this procedure, calcium chloride is present in the reaction solution at approximately 3 mmol/L or less. However, it is not disclosed nor suggested that a high concentration should be maintained in the reaction solution for the immunological measurement of cardiac troponin, and that an accurate value sometimes cannot be obtained at a low concentration (for example, approximately 3 mmol/L) depending on the type of specimen. In connection with this, the calcium chloride concentration in the troponin assay buffer used in the Examples of patent-literature 3 is 2 mmol/L (for example, Examples 10 to 12).
Patent literature 4 discloses that the concentrations of calcium ions and magnesium ions are not important as buffers used in the preparation of a troponin complex, but it should be preferably about 20 μmol/L to about 20 mmol/L, and the typical amount of calcium and/or magnesium is about 2 to 5 mmol/L. Although it is not clearly described, it is disclosed in Examples 1 and 2 that about 225 mg/L calcium chloride or other calcium salts capable of providing 1 to 3 mmol/L calcium ions are contained in the reaction solution for the preparation of a complex. However, patent literature 4 does not refer to the concentration of calcium chloride or magnesium chloride in a reaction solution during the troponin assay. Further, it is not disclosed nor suggested that a high concentration should be maintained in the reaction solution for the immunological measurement of cardiac troponin, and that an accurate value sometimes cannot be obtained at a low concentration (for example, approximately 3 mmol/L) depending on the type of specimen.
As described above, patent literatures 2, 3, and 4 disclose that calcium chloride or magnesium chloride is generally added at a concentration of about 1 to 6 mmol/L for the stabilization of a sample containing troponin complexes, and it is not intended to improve the reliability of the measured value of an immunological reaction.